Enzyme chemically coupled to cellulose ether

ABSTRACT

A process for the preparation of a water insoluble papain which process comprises reacting at 0*-3*C. the papain dissolved in a buffer within a pH range of 6.5-8.5 and containing L-cysteine and diaminoethane tetra-acetic acid with the p-diazophenoxy hydroxypropyl ether of cellulose.

United States Patent [191 Emery 1 Jan.30, 1973 [75] inventor: AntohnyNicholas Emery, Birmingham, Warwickshire, England [73] Assignee: RanksHovis McDougall Limited,

London,England 221 Filed: March 17, 1970 21 Appl. No.: 20,389 1 [30]Foreign Application Priority Data Dec. 11, 1969 Great Britain..60,474/69 [52] U.S. Cl. ..195/63, 99/48,195/68, l95/D1G. 11

[51] Int. Cl ..C07g 7/02, C12n 1/00 [58] Field of Search ..195/63, 63 P,68; 99/48 [56] References Cited UNITED STATES PATENTS 3,597,219 8/1971Wildi et a1. ..99/48 3,167,485 1/1965 Katchalski et al ..195/633,117,004 1/1964 'McFarlane et al. ..99/48 3,282,702 11/1966 Schreiner..195/63 X OTHER PUBLlCATlONS Barker et al., Preparation and Propertiesof a-Amylase Chemically Coupled to Microcrystalline Cellulose.Carbohydrate Research. Vol. 8, 1968 (pp. 491497) QD321C3.

Dixon, et a1., Enzymes, Academic Press Inc, N.Y. 2nd Ed. 1964 (p. 346)QP601.D5EC.2.

Silman, et al., Water-Insoluble Derivatives of Enzymes, Antigens andAntibodies Annual Review of Biochemistry Vol. 35 Part 11 1966 (pp.881886) QP501A7.

Primary Examiner-Lionel M. Shapiro Assistant Examiner-David M. NaffAttorney-Stevens, Davis, Miller & Mosher [57] ABSTRACT A process for thepreparation of a water insoluble papain which process comprises reactingat 03C. the papain dissolved in a buffer within a pH range of 6.5-8.5and containing L-cysteine and diaminoethane tetra-acetic acid with thep-diazophenoxy hydroxypropyl ether of cellulose.

6 Claims, No Drawings enzymes ENZYME CHEMICALLY COUPLED TO CELLULOSEETHER It is well known that when an enzyme is attached toan insolublesupport the novel micro-environment of the enzyme markedly affects itsstability. Hydrophilic features of the carrier tend to enhance thestability of the attached enzyme whereas hydrophobic features have theopposite effect. Polysaccharide carriers such as fibrous cellulose (M.A.Mitz and L.J. Summaria, Nature, London, 189, (1961), 576 & W.E.l-lornby, M.D., Lilly and EM. Crook, Biochem, J., 98, (1966), 420) andcross-linked dextran (R. Axen and J.- Porath, Nature, London, 210,(1966), 367) have been shown to be particularly effective in conferringstability to the attached enzyme.

Commercial samples of water insoluble forms of trypsin,chymotrypsin,'ribonuclease, glucose oxidase and ficin became availablein February 1968. These were obtained by reaction of the appropriateenzyme with carboxymethyl cellulose hydrazide and are marketed by.Seravac Laboratories, Ltd., Maidenhead, Berks.

Almost simultaneously (January 1968) Miles Laboratories, Inc., Elkhart,U.S.A., marketed water insoluble forms of trypsin, chymotrypsin andpapain in which the enzymes were bound to ethylene-maleic anhydridecopolymer carrier.

R. Axen and J. Porath, Nature 210 (1966), 367., succeeded in preparingactive water insoluble chymotryp sin and trypsin by reaction of theenzymes with pisothiocyanato phenoxy hydroxypropyl Sephadex(cross-linked dextran).

Our copending British application No. 14074/69 provided active waterinsoluble preparations of other 1.e., dextranase wherein the enzyme ischemically coupled with the p-diazophenoxyhydroxypropyl ether derivedfrom microcrystalline cellulose.

It is an object of the present invention to provide an activewater-insoluble preparation of papain wherein the enzyme is chemicallycoupled with the P- diazophenoxyhydroxypropyl ether derived frommicrocrystalline cellulose.

The present invention provides a water insoluble papain chemicallycoupled to p-diazophenoxyhydroxypropyl cellulose.

According to the present invention there is provided a process for thepreparation of a water insoluble papain which process comprises reactingat 0-3C. the papain dissolved in a buffer within a pH range 6.5-8.5 andcontaining L-cysteine and diaminoethane tetraacetic acid with thep-diazophenoxy hydroxypropyl ether of cellulose.

A water insoluble preparation of papain may be made by chemical reactionof the papain dissolved in a buffer within a pH range of 6.5-8.5(preferably pullulanase, carboxypeptidase and phosphate buffer 0.075M,pH 6.9) containing L- cysteine, 5mM., and diaminoethane tetra-aceticacid, 2mM. Unreacted diazo groups in the cellulose derivative can beannealed by reaction with B-naphthol or phenol. Preferablymicro-crystalline cellulose is used for the preparation of this ether.An active water-insoluble papain can be obtained by this process whichis more heat stable when suspended in an aqueous buffer (0.02M) than thecorresponding soluble enzyme. Preferably the buffer should have that pHat which the enzyme displays maximum enzymic activity towards itsubstrate.

The particular merits of the present invention for providingwater-insoluble papain is that it can provide a product with a highretention of activity when calculated as a percentage of the activitywhich that amount of enzyme protein bound to the cellulose derivativewould display in its original soluble form. The second advantage is thatthe use of micro-crystalline cellulose in the preparation of the etheraffords a dense hydrophilic carrier available in a fine particulate formfor maximum surface exposure yet easily recoverable after use bycentrifugation or filtration and which is then suitable for reuse.Alternatively the water-insoluble enzyme may be used to treat, forexample, Beer, on a continuous basis for an extended period of time. Thethird advantage is the much greater heat stability of the waterinsoluble enzyme giving a greater shelf life and enabling the enzyme tobe used at a higher operating temperature and for a longer period oftime.

Following is a description by way of example of methods of carrying theinvention into effect:

EXAMPLE I Samples mg.) of p-amino phenoxy hydroxypropyl cellulose ether(prepared according to the method of Barker, S.A., Somers, P..l., andEpton, R., Carbohydrate Research8, 491, (1968)) were placed in acentrifuge tube. Aliquots (5 ml.) of ice-cold, lN hydrochloric acid wereadded and the slurry stirred magnetically for 15 minutes at 0C. Aliquots(4 ml.) of ice-cold 2 percent sodium nitrite solution were added andafter a further 15 minutes the tubes were centrifuged and thesupernatant discarded. The solid was washed 3 times with aliquots (15ml.) of phosphate buffer (0.075M pH 6.5-8.5). After the final washingshad been decanted, aliquots (1 ml.) of a solution of papain (10 mg/mlactivity 0.148 units/mg protein) in phosphate buffer (0.075M pH 6.58.5)containing L- cysteine (SmM) and EDTA (diaminoethane tetraacetic acid)(2mM), were added and the tubes stirred magnetically at 0-3C. for 48hours. After coupling, 5 ml. of an ice-cold 0.01 percent solution ofB-naphthol in saturated aqueous sodium acetate were added. After afurther 15 minutes stirring, the water insoluble papain derivatives weresubjected to 5 cycles of washing with phosphate buffer (0.03M pH 6.9 15ml.) and sodium chloride solution (0.5M) in the same buffer. The papainderivatives were finally washed twice with phosphate buffer (0.02M pH6.9).

The activity of the water insoluble papain was determined in thefollowing manner. Samples were suspended (or dissolved in the case of'free papain) in phosphate buffer (5 ml. 0.075M pH 6.5). 5 Ml. ofactivator solution, (L-cysteine 5mM and'EDTA 2mM) RESULTS Prep. pH ofNo.

. Enzyme units/ coupling mg bound protein mg bound Enzyme protein peractivity 100 m retention derivative EXAMPLE II A sample ofwater-insoluble papain (10 mg.) was prepared according to Example I. Thecoupling was carried out at pH 6.9 and gave a derivative containing 0.81mg. protein per 100 mg. derivative and with a specific activity equal to0.041 enzyme units/mg protein. The sample was suspended in phosphatebuffer (0.02M pH 6.9, 5 ml.), mixed with an activator solution (5 ml.containing L-cysteine 5mM and EDTA 2mM) and incubated with ml. of apre-warmed 2 percent casein solution at 50C. for 1 hour in amagnetically stirred test tube, after which the samples were rapidlycooled and the papain activity assayed at 30C.

A control incubation was performed in which the water insoluble papainwas replaced by a solution of an equivalent amount of free papain inphosphate buffer (0.02M pH 6.9 5 ml.) and activator solution (5 ml.).

Results Prep. No.

% retention of activity Final activity O. D. units/ mm. 0.0030

Initial activity O.D. units/ min.

diazo 4 coupled Free EXAMPLE III A sample of water insoluble papain wasprepared as in Example 1. The coupling was carried out at pH 6.9 andgave a derivative containing 0.809 mg. protein/100 mg. carrier and witha specific activity of 0.105 enzyme units per mg. ofprotein.

1 g. of the water insoluble papain derivative was placed in a plastic 1liter centrifuge pot. Two 1 pint bottles of Beer (original gravity1.035, fermented for 5 days, chilled and filtered) were opened andpoured into the centrifuge pot. Potassium metabisulphite was added (50mg/l.) and the Beer was then magnetically stirred at room temperaturefor 72 hours. The pot was centrifuged for minutes at 1,100 g., the Beerdecanted back lnto bottles, which were crowned and repasteurized.Further bottles were opened and treated in the same way for periods of24 hours and 18 hours using the same solid. After the final treatmentthe water-insoluble papain was washed in distilled water and resuspendedin phosphate buffer (0.02M pH 6.9 ml.). An aliquot (5 ml.) of thesuspension was assayed as before.

The bottles of treated Beer, together with similar but untreatedcontrols were stored for alternate periods at 4C. and room temperatureto accelerate the development of haze. Haze readings were taken atintervals.

lliESULTS Prep. Activity of papain Activity No. (O.D. units/min.Retention 5 Initially 8.9 X 10" 5 After 10 4.7 X 10' 53 days treatmentHaze (E.B.C. Units) Time of treatment Days of incubation 0 12 14 15 72hours 2.5 3.3 2.5 2.3 24 hours 2.5 6.3 8.8 9.3 18 hours 2.5 6.4 9.7 10.5CONTROL 2.5 9.8 13.0 13

EXAMPLE 1V Samples 10 mg.) of water insoluble papain (preparation No.4,0.81 mg. protein/mg solid derivative) were suspended in phosphate buffer(5 ml., 0.075M pH 6.5). Dilutions of casein (0.032-0.8mM) were preparedin phosphate buffer (5 ml. 0.075M pH 6.5 taking a 1 percent caseinsolution as equivalent to 0.4mM. The velocity of the initial reaction ofthe water insoluble papain on casein for each dilution was determined bythe method in Example 1.

RESULTS lnitial casein initial Reaction concentration Velocity in OD.units/min. 0.032 0.0012

1 claim:

1. A process for the preparation of a water insoluble enzymaticallyactive papain which process comprises reacting at 03C. the papaindissolved in a buffer within a pH range of 6.5-8.5 and containingL-cysteine and diaminoethane tetra-acetic acid with the pdiazophenoxyhydroxypropyl ether of cellulose.

2. A process as claimed in claim 1 wherein the pH is between 6.8 and7.0.

3. A process as claimed in claim 1 wherein unreacted diazo groups in thecellulose derivative are annealed by reaction with B-naphthol or phenol.

'4. A process as claimed in claim 1 wherein microcrystalline celluloseis used for the preparation of the ether.

5. Enzymatically active papain chemically coupled top-diazophenoxyhydroxypropyl cellulose.

6. A process as defined in claim 1, wherein the pH is between 6.5 and7.0.

1. A process for the preparation of a water insoluble enzymaticallyactive papain which process comprises reacting at 0*-3*C. the papaindissolved in a buffer within a pH range of 6.5-8.5 and containingL-cysteine and diaminoethane tetra-acetic acid with the p-diazophenoxyhydroxypropyl ether of cellulose.
 2. A process as claimed in claim 1wherein the pH is between 6.8 and 7.0.
 3. A process as claimed in claim1 wherein unreacted diazo groups in the cellulose derivative areannealed by reaction with Beta -naphthol or phenol.
 4. A process asclaimed in claim 1 wherein microcrystalline cellulose is used for thepreparation of the ether.
 5. Enzymatically active papain chemicallycoupled to p-diazophenoxyhydroxypropyl cellulose.